Rev-Erb is a fascinating orphan nuclear receptor encoded on the opposite strand of a thyroid hormone receptor gene. Rev-Erb belongs to a subset of the thyroid/retinoid receptor superfamily that includes ROR-alpha, which is crucial for cerebellar development. Rev-Erb and ROR-alpha are distinguished from other nuclear receptors because both bind DNA as monomers, requiring a receptor half-site flanked 5 prime by an extended A/T rich region. Rev-Erb and ROR-alpha are functionally antagonistic; Rev-Erb is a repressor whereas ROR-alpha is a transcriptional activator. Rev-Erb only represses transcription as a homodimer on a direct repeat of the monomer binding site spaced by 2 bp, to which it binds cooperatively. The first specific aim of this proposal is to understand the determinants of DNA binding by Rev-Erb and ROR-alpha. The roles of the extended DNA-binding domain will be delineated biochemically and by crystallographic analysis of purfied Rev-Erb bound to monomer and dimer sites. The molecular basis for homodimerization and lack of heterodimerization with RXR will be determined by analysis of receptor chimera and mutations. The second specific aim is to compare and contrast the transcriptional regulatory properties of ROR-alpha and Rev-Erb. Preliminary results suggest that transactivation by ROR-alpha requires dimerization, and the hypothesis that DNA-binding regulates interaction between ROR- alpha and transcriptional cofactors will be tested. In addition, an inherent, cell-specific repression domain in ROR-alpha will be characterized. The third specific aim is to characterize the mechanism and biological function of Rev-Erb and ROR-alpha interactions with the nuclear hormone receptor corepressors N- CoR and SMRT. Preliminary results indicate that N-CoR interacts differentially with Rev-Erb on different binding sites. Furthermore, different regions of Rev-Erb govern binding to N- CoR versus SMRT. ROR-alpha also interacts with N-CoR and SMRT. The molecular basis and function of these findings will be determined. The fourth specific aim is to characterize the mechanism and function of a novel Rev-Erb corepressor that we have isolated using the yeast two-hybrid system. The molecular basis of the interaction will be determined, and the biological relevance of the novel corepressor will be explored. Together, these studies will provide insight into the molecular mechanisms by which members of the Rev-Erb and ROR-alpha interact with target genes and coregulator proteins to both positively and negatively regulate transcription. These studies are required to understand the function of Rev-Erb and ROR-alpha and, more generally, will define novel mechanisms underlying specificity of gene regulation by the nuclear hormone receptor superfamily.